The Major Histocompatibility Complex (MHC) represents the most important susceptibility locus for many common human autoimmune diseases, including type 1 diabetes, rheumatoid arthritis and multiple sclerosis, indicating that antigen presentation to CD4 T cells represents an important step in their pathogenesis. HLA-DM (DM) plays a central role in the MHC class II (MHCII) antigen presentation pathway: it induces dissociation of the invariant chain-derived CLIP peptide and edits the peptide repertoire, favoring presentation of high-affinity peptides. The central goals of this project are to define th mechanism of DM action at a structural level and to advance our understanding of self-antigen presentation in autoimmune diseases. Preliminary studies have shown that the interaction of DM and HLA-DR (DR) molecules is controlled by the occupancy state of the DR peptide binding groove. We found that DM cannot bind to DR proteins when the peptide binding groove is fully occupied by peptides. Rather, DM binds to an unstable transition state of DR proteins in which the peptide N-terminus has partially dissociated from the groove. We utilized this insight to develop a strategy for crystallization of the complex of DM bound to DR1. Aim 1 Since submission of the original proposal, we have been able to determine the crystal structure of the DR1-DM complex at a resolution of 2.6. The structure suggests a novel mechanism for rapid selection of high-affinity peptides (also referred to as 'editing'). In the DM-bound state, three D residues have moved into a critical part of the groove (P1 pocket and P2 site), rendering it initially inaccessible to peptides. Peptides need to compete for access to these sites, and this energetic barrier drives selection of peptides with the highest affinities. We have also recently developed a novel approach to measure rapid binding of peptides to empty DR1-DM complexes. This technique will now be used to define critical mechanistic steps in editing of the peptide repertoire by DM. Aim 2 is based on novel observations from the DR1-DM structure. HLA-DQ8 (DQ8) confers susceptibility to type 1 diabetes while HLA-DQ6 (DQ6) induces dominant protection. The DR1-DM structure shows that a substantial number of residues located at the DR1-DM interface are polymorphic in DQ8 and DQ6. Furthermore, preliminary data show substantial differences in the peptide binding properties of DQ8 and DQ6. We will use a series of functional approaches to dissect the impact of these DQ polymorphisms on peptide presentation by DQ8 and DQ6. Also, the novel strategy that enabled crystallization of the DR1-DM complex will be used for crystallization trials of a DQ-DM complex. We anticipate that these studies will have a significant impact on our understanding of DM function and MHCII antigen presentation in autoimmune diseases.